The use of the PMI/mannose selection system to recover transgenic sweet orange plants ( Citrus sinensis L. Osbeck)

Abstract

A new method for obtaining transgenic sweet orange plants was developed in which positive selection (Positech) based on the Escherichia coli phosphomannose-isomerase (PMI) gene as the selectable marker gene and mannose as the selective agent was used. Epicotyl segments from in vitro-germinated plants of Valencia, Hamlin, Natal and Pera sweet oranges were inoculated with Agrobacterium tumefaciens EHA101-pNOV2116 and subsequently selected on medium supplemented with different concentrations of mannose or with a combination of mannose and sucrose as a carbon source. Genetic transformation was confirmed by PCR and Southern blot. The transgene expression was evaluated using a chlorophenol red assay and isoenzymes. The transformation efficiency rate ranged from 3% to 23.8%, depending on cultivar. This system provides an efficient manner for selecting transgenic sweet orange plants without using antibiotics or herbicides.

Expression of The Human Acidic Fibroblast Growth Factor in Transgenic Tomato and Safety Assessment of Transgenic Lines

“…The effective mannose concentration varied among species, ranging from 0.36 g/l for Arabidopsis (63) to 10-50 g/l for rice (39). Our results confirmed the published data that better transformation efficiency is achieved when lower selection stringency of mannose is combined with a low supplemental carbohydrate concentration (5,72). Therefore, we used a combination of 10 g/l mannose and 5 g/l glucose during the first 6 weeks of the selection.…”

Transformação genética de laranja doce (Citrus sinensis L. Osbeck) com o gene D4E1 dirigido pelos promotores CaMV35S ou AtPP2

“…Estes resultados de eficiência estão de acordo com os resultados de outros trabalhos, em que a eficiência de transformação utilizando-se epicótilos de laranja "Pêra" foi menor independente da construção gênica utilizada na transformação (AZEVEDO, 2005;BARBOSA-MENDES, 2007;BOSCARIOL, 2004), comprovando que a eficiência de transformação varia com o genótipo e também é influenciada pela construção gênica utilizada, pela habilidade morfogênica e pela afinidade do genótipo com a Agrobacterium tumefaciens GHORBEL et al, 2000;MIYATA et al, 2011 (2005), de 2,5 a 3% para laranja "Hamlin", 0,5% para laranja doce "Pêra" e 3,3 a 4,5% para laranja "Valência". A eficiência de transformação sem gene repórter no trabalho realizado por Barbosa-Mendes (2007), utilizando-se como explantes epicótilos de laranja doce foi de 6,2% para laranja "Hamlin", 2,7% para laranja "Pêra" e 5,6% para laranja "Valência".…”

Recent Advances in Fruit Species Transformation

“…In Citrus sinensis, the best results were obtained when 13 g/L mannose as unique source of carbon was added into the selection media. Mannose combined with other sugars promoted reduction in transformation efficiencies and escapes (Boscariol et al, 2003). Apricot cv.…”